Generation of Highly Functional Hepatocyte-like Organoids from Human Adipose-Derived Mesenchymal Stem Cells Cultured with Endothelial Cells.

Previously, we successfully established a highly functional, three-dimensional hepatocyte-like cell (3D-HLC) model from adipose-derived mesenchymal stem cells (ADSCs) via a three-step differentiation protocol. The aim of the present study was to investigate whether generating hepatocyte-like organoids (H-organoids) by adding endothelial cells further improved the liver-like functionality of 3D-HLCs and to assess H-organoids' immunogenicity properties. Genes representing liver maturation and function were detected by quantitative reverse transcription-PCR analysis. The expression of hepatic maturation proteins was measured using immunofluorescence staining. Cytochrome P (CYP)450 metabolism activity and ammonia metabolism tests were used to assess liver function. H-organoids were successfully established by adding human umbilical vein endothelial cells at the beginning of the definitive endoderm stage in our 3D differentiation protocol. The gene expression of alpha-1 antitrypsin, carbamoyl-phosphate synthase 1, and apolipoprotein E, which represent liver maturation state and function, was higher in H-organoids than non-organoid 3D-HLCs. H-organoids possessed higher CYP3A4 metabolism activity and comparable ammonia metabolism capacity than 3D-HLCs. Moreover, although H-organoids expressed human leukocyte antigen class I, they expressed little human leukocyte antigen class II, cluster of differentiation (CD)40, CD80, CD86, and programmed cell death ligand 1, suggesting their immunogenicity properties were not significantly upregulated during differentiation from ADSCs. In conclusion, we successfully established an H-organoid model with higher liver-like functionality than previously established 3D-HLCs and comparable immunogenicity to ADSCs.